Magnetic resonance imaging (MRI) is a technique that has become particularly attractive to physicians as images of a patient's body or parts thereof can be obtained in a non-invasive way and without exposing the patient and the medical personnel to a potentially harmful radiation such as X-rays. Because of its high quality images and good spatial and temporal resolution. MRI is a favourable imaging technique for imaging soft tissue and organs. MRI may be carried out with or without MR contrast agents. However, contrast-enhanced MRI usually enables the detection of much smaller tissue changes, which makes it a powerful tool for the detection of early stage tissue changes like for instance small tumors or metastases.
MRI using hyperpolarized molecules is an emerging technique. WO 9935508 discloses a method of MR investigation of a patient using a hyperpolarized solution of a high T1 agent as MRI contrast agent. The term “hyperpolarization” means enhancing the nuclear polarization of the NMR active nuclei present in the agent, i.e. nuclei with non-zero nuclear spin, preferably 13C- or 15N-nuclei, and thereby amplifying the MR signal intensity by a factor of hundred and more. When using a hyperpolarized 13C- and/or 15N-enriched high T1 agent, there will be essentially no interference from background signals as the natural abundance of 13C and/or 15N is negligible and thus the image contrast will be advantageously high. The main difference between conventional MRI contrast agents and these hyperpolarized high T1 agents is that in the former changes in contrast are caused by affecting the relaxation times of water protons in the body whereas the latter class of agents can be regarded as non-radioactive tracers, as the signal obtained arises solely from the agent. When hyperpolarization is obtained via a microwave assisted transfer between unpaired electrons and the nuclei used as MR probes, the techniques is referred as Dynamic Nuclear Polarization (DNP).
A variety of possible high T1 agents for use as MR imaging agents are disclosed in WO9935508, including non-endogenous and endogenous compounds. As examples of the latter, intermediates in normal metabolic cycles are mentioned which are said to be preferred for imaging metabolic activity. By in vivo imaging of metabolic activity, information of the metabolic status of a tissue may be obtained and said information may for instance be used to discriminate between healthy and diseased tissue.
For example, WO 2009077575 discloses a method of 13C-MR detection using an imaging medium comprising hyperpolarized 13C-fumarate, in order to investigate both the citric acid and the urea cycles by detecting 13C-malate end optionally 13C-fumarate and/or 13C-succinate signals. The metabolic profile generated in a preferred embodiment of the method provides information about the metabolic activity of the body and part of the body under examination and said information may be used in a subsequent step for. e.g. identifying diseases. Such a disease is preferably cancer since tumor tissue is usually characterized by an altered metabolic activity. As a technical aspect, if the compounds to be polarized crystallize upon freezing or cooling their solution, a glass-forming additive must be added to the solution.
Dynamic nuclear polarization (DNP) has been applied recently to magnetic resonance spectroscopy (MRS) in solution, where it can be used to produce a large increase in sensitivity. Using this technique, the metabolism of several 13C-labeled compounds has been observed and used to estimate rate constants for specific enzyme-catalyzed reactions in vitro and in vivo (Day S E, Kettunen M I, Gallagher F A, Hu D E, Lerche M, Wolber J, Golman K, Ardenkjaer-Larsen J H, Brindle K M. Detecting tumor response to treatment using hyperpolerized 13C magnetic resonance imaging and spectroscopy. Nat Med 2007; 13:1382-1387; Gallagher F A, Kettunen M I, Hu D E, Jensen P R, Zandt R I, Karisson M, Gissetsson A, Nelson S K, Witney T H, Bohndiek S E, Hansson G, Peitersen T, Lerche M H, Brindle K M. Production of hyperpolarized [1.4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors. Proc Nag Acad Sci USA 2009; 106:19801-19806). Furthermore, for some hyperpolarized 13C-labeled substrates there is sufficient signal for the spatial distribution of both the substrate and its metabolites to be imaged in vivo. As some of these substrates have already been administered at relatively high concentrations in the clinic, this technique has the potential to be translated into clinical applications. To date, the most studied reactions have been those involving hyperpolarized [1-13C]pyruvate: the hyperpolarized label can be exchanged with either endogenous lactate or alanine, or alternatively it can be irreversibly converted to carbon dioxide, which is subsequently converted to bicarbonate in the reaction catalyzed by carbonic anhydrase. These metabolic reactions have been observed in tumors, in cardiac tissue and in the liver (Merritt M E, Harrison C, Storey C, Jeffrey F M, Sherry A D, Malloy C R. Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR. Proc Natl Acad Sci USA 2007; 104:19773-19777; Schroeder M A, Swietach P, Atherton H J, Gallagher F A, Lee P, Radda G K, Clarke K, Tyler D J. Measuring intracellular pH in the heart using hyperpolarized carbon dioxide and bicarbonate: a 13C and 31P MRS study. Cardiovasc Res 2010; 86:82-91: Hu S, Chen A P, Zierhut M L, Bok R, Yen Y F, Schroeder M A, Hurd R E, Nelson S J, Kurhanewicz J, Vigneron D B. In vivo carbon-13 dynamic MRS and MRSI of normal and fasted rat liver with hyperpolarized 13C-pyruvate. Mol Imaging Bid 2009; 11:399-407).
Recently, other endogenous molecules have been successfully hyperpolarized: tumor pH has been measured in vivo from the relative concentrations of 13C-labeled bicarbonate and carbon dioxide following the injection of hyperpolarized 13C-labeled bicarbonate (Gallagher F A, Kettunen M I, Day S E, Hu D E, Ardenkjaer-Larsen J H, Zandt R, Jensen P R, Karlsson M, Golman K, Lerche M H, Brindle K M. Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate. Nature 2008; 453:940-943); elevated levels of hyperpolarized malate have been demonstrated in necrotic tumor tissue in vivo following the injection of hyperpolarized 13C-labeled fumarate (Gallagher F A, Kettunen M I, Hu D E, Jensen P R, Zandt R I, Karisson M, Gisselsson A, Nelson S K, Witney T H, Bohndiek S E, Hanason G, Peitersen T, Lerche M H, Brindle K M. Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors. Proc Natl Acad Sci USA 2009; 106:19801-19806); the metabolism of glutamine to glutamate, catalyzed by the mitochondrial enzyme glutaminase, has been observed following administration of hyperpolarized 13C-labeled glutamine to cells in vitro (Gallagher F A, Kettunen M I, Day S E, Lerche M, Brindle K M. 13C MR spectroscopy measurements of glutaminase activity in human hepatocellular carcinoma cells using hyperpolarized 13C-labeled glutamine. Magn Reson Med 2008; 60:253-257); the organ-specific metabolism of hyperpolarized 13C-labeled acetate to acetyl-CoA and acetyl carnitine has been observed in vivo (Jensen P R, Peitersen T, Karlsson M, In't Zandt R, Gisseisson A, Hansson G, Meier S, Lerche M H. Tissue-specific short chain fatty acid metabolism and slow metabolic recovery after ischemia from hyperpolarized NMR in vivo. J Biol Chem 2009:284:36077-36082), and the metabolism of branched chain amino acids has been observed in tumors following the addition of hyperpolarized 13C-labeled α-ketoisocaproate (Karisson M, Jensen P R, In't Zandt R, Gisselsson A, Hansson G, Duus J O, Meier S, Lerche M H. Imaging of branched chain amino acid metabolism in tumors with hyperpolarized 13C ketoisocaproate. Int J Cancer 2010:127:729-736.10).
Although its etiology is lacking, cancer is phenomenologically well characterized as a molecular disease. Different kinds of cancers may have very different biochemical forms, however they can share general molecular features.
Early diagnosis of cancer continues to be given large attention since diagnosis at an early stage often increases the chances of a successful treatment. In fact, early diagnosing cancer and ensuring access to optimum treatment can lead to significant improvements in survival.
Early diagnosing of cancer could be achieved by taking advantage of a general molecular feature shared by different types of cancer cells and whose alteration in cancer can be early detected.
Carboxylesterases (CE, EC 3.1.1.1) are a family of enzymes catalysing the chemical conversion of an ester in an acid and an alcohol. A general reaction scheme is shown below:

Carboxylesterases are ubiquitously expressed in mammalian tissues. The many CE isoforms have been classified into 5 super families (CE 1-5) based on amino acid homologies. The CE1 enzymes are mainly localized to the liver, however they are also expressed in most other tissue types. A rat specific CE1 isoform is secreted from the liver to the blood in rats and mice and this iso-enzyme is correlated to a high level of hydrolase activity detected in rodents compared to humans (Yan, B. Dongfang Y., Bullock, P., Parkinson, A., Rat serum carboxylesterases, 1995, JBC, 32 (270): 19128-34; Rudakova, E V., Botneva N P., Makhaeva, G F. Comparative analysis of esterase activities of human, mouse and rat blood, 2011, Bulletin of experimental biology and medicine, 152(1): 73-75). The other important isoform is the CE2 family, which is also expressed in the human liver (approx. 4 times less than CE1) as well as in most other tissues to a higher degree than CE1 (Talvar, S. The expression of human carboxylesterases in normal tissues and cancer cell lines (2008), Master thesis).
The expression of carboxylesterases decreases in cancer in both animal and human tissue. In particular, in hepatome cells a 4 times decrease in the expression of carboxyl esterase has been measured compared to normal hepatocytes. Dependent on the isoform the expression is reported to be approx. 1.5-4 times higher in normal tissue than in the corresponding malignant tissue (Talvar, 2008).
A number of studies have been reported on carboxylesterases in cancer cells.
The expression of carboxylesterase was reported as detectable in human cancer cells (HEPG2) and approx. 3-4 times lower than the expression of carboxyl esterase in normal human liver (hepatocytes) (Talvar, 2008). A patient study on non-tumor and tumor tissues from liver cancer (HCC) patients showed an almost 3 times decrease in the expression of carboxyl esterase in the tumor tissue (Na, K. et al., Human plasma carboxylesterase 1, a novel serologic biomarker candidate for hepatocellular carcinoma (2009). Proteomics, 9: 3989-99).
Another study showed that the carboxylesterase activity was significantly lower in colon cancer xenografts compared to the corresponding normal colon tissue in mice (Jansen et al., CPT-11 In human colon cancer cell lines and xenografts: characterization of cellular sensitivity determinants, 1997, Int. J. Cancer 70:335-40.)
A study has been reported on lung cancer patients where carboxylesterase activity is correlated to esterase expression in healthy and cancer lung tissue. In this study, they find that the activity correlates well with the expression, which is shown to be approx. 1.5 times higher in healthy tissue (Liewald F. et al, Intracellular pH, esterase activity and DNA measurements of human lung carcinomas by low cytometry, 1990, Cytometry, 11: 341-48)
WO2012102773 discloses a method for the diagnosis and treatment of cancer, in particular breast cancer, by measuring the activity of the enzyme PMPMEase (human carboxylesterase 1). Said activity is measured in a biological sample by assaying the enzyme expression or enzymatic activity, in the last case through the measurement of the consumption of a substrate or the production of a product. It is only generally stated that the enzyme assay can be performed in vivo.
U.S. Pat. No. 8,198,038 discloses a screening method to distinguish healthy human beings from those with human liver cancer (hepatocellular carcinoma: HCC) comprising the steps of collecting human blood and detecting the presence of human liver carboxylesterase 1 (hCE1) in the plasma, wherein the level of hCE1 protein is increased more in the plasma of patients with HCC than in the plasma of healthy patents.
The conversion of an hyperpolarized ester, catalysed by carboxylesterase, generates hyperpolarized metabolic products whose MR signals are well distinguishable from each other and from the injected substrate. Cancer cells and healthy cells convert a hyperpolarized ester to a different degree, leading to differences in said metabolic product signal amounts, therefore said difference between the signals in tumor and non-tumor cells can be exploited to identify cancer.
A common weakness of the endogenous molecules known in the prior art, when used as hyperpolarized markers, is the relatively fast vanishing of MR signals, of both the substrate itself and its relevant metabolites. DNP in fact, as well as all other hyperpolarization procedures, leads to a transient enhancement of the nuclear magnetic order, that allows the in vivo visualization of said moieties but, unfortunately, for a limited amount of time, in the order of 3 to 5 times the longitudinal relaxation time of the nuclei used as MR probe. Since MR imaging procedures require a certain amount of time in order for the final image to be reconstructed with acceptable sensitivity and resolution, markers with slower nuclear relaxation, with respect to the product used so far, are strongly desirable, also in view of inherent technical features of the imaging procedure, such as dilution (upon injection into patient) and metabolic conversion (time delay for achieving detectable concentrations of metabolites).
Therefore, in order to be able to register in vivo metabolic maps of tumour and healthy tissues, with superior sensitivity and resolution with respect to the state of the art, novel metabolic contrast agents with enhanced persistence of the hyperpolarized signal (i.e. slower nuclear longitudinal relaxation) are strongly needed.